Tick-borne human disease cases are rising and outnumber mosquito-borne diseases in temperate climates. In North America, Ixodes scapularis ranks as the tick of highest public health concern as vector of Borrelia burgdorferi (Bb, an agent of Lyme disease), Anaplasma phagocytophilum (Ap, agent of human anaplasmosis), and Powassan or deer tick virus (agent of Powassan encephalitis), among others. In this resubmission, we will take advantage of our advances in rickettsial genomics to create rickettsiae able to express transgenes in ticks. Transgenic approaches are not yet available for ticks, in part hindered by their months- to year-long generation times. Our ability to generate aposymbiotic ticks coupled with our membrane feeder to reconstitute ticks with transformed R. buchneri and challenge them experimentally with Ap and Bb has opened unprecedented possibilities to dissect the interactions between ticks, their symbionts, and the pathogens they transmit. Symbionts play a fundamental role in shaping an arthropod's immune system. This highlights the need to understand how the tick immune system works when investigating the possibility of a paratransgenic strategy for tick-borne disease control. The tick immune system is not well understood, but Dr. Pedra, a co-investigator in the renewal project, has discovered fundamental differences from the insect immune system. The overarching hypotheses of our proposal are 1) that the rickettsial symbiont of I. scapularis (Rickettsia buchneri) plays a core role in regulating this tick's relationship with pathogens that determines its capacity to be a vector, and 2) that the tick immune system is an important intermediary in this triad. In order to dissect the interacting components, we will address the following aims: Aim 1) Determine R. buchneri role in tick fitness, exclusion of other rickettsiae, susceptibility to Ap and Bb. Aim 2) Prepare R. buchneri transposon mutants that are competent to colonize ticks using a Himar1 transposon designed for subsequent replacement using Recombinase Mediated Cassette Exchange (RMCE). Aim 3) Replace the Himar1 transposon with an expression cassette encoding an anti-Ap Nanobody in colonization competent R. buchneri mutants; determine susceptibility of reconstituted I. scapularis to Ap. Aim 4.1) Determine whether R. buchneri presence in I. scapularis affects the activity of the IMD pathway. Aim 4.2) Identify host factors that alter immune signaling during colonization of I. scapularis by Ap and Bb in the presence or absence of R. buchneri. At the conclusion of the proposed project we will have gained detailed knowledge about R. buchneri as a provider of essential nutrients to ticks, as a factor required for maturation of a functional tick immune system, and as a controller of pathogen acquisition and transmission. We will further have defined the conditions that must be met to introduce a genetically modified R. buchneri into I. scapularis so that an effective paratransgenesis approach could be devised.